Plate-like Guanine Biocrystals Form via Templated Nucleation of Crystal Leaflets on Preassembled Scaffolds

Controlling the morphology of crystalline materials is challenging, as crystals have a strong tendency toward thermodynamically stable structures. Yet, organisms form crystals with distinct morphologies, such as the plate-like guanine crystals produced by many terrestrial and aquatic species for light manipulation. Regulation of crystal morphogenesis was hypothesized to entail physical growth restriction by the surrounding membrane, combined with fine-tuned interactions between organic molecules and the growing crystal. Using cryo-electron tomography of developing zebrafish larvae, we found that guanine crystals form via templated nucleation of thin leaflets on preassembled scaffolds made of 20-nm-thick amyloid fibers. These leaflets then merge and coalesce into a single plate-like crystal. Our findings shed light on the biological regulation of crystal morphogenesis, which determines their optical properties.

Chemical fixation and conventional TEM. Samples were fixed with 4% paraformaldehyde, 2% glutaraldehyde in 0.1 M cacodylate buffer containing 5 mM CaCl2 (pH 7.4) for 1 hr, postfixed in 1% osmium tetroxide supplemented with 0.5% potassium hexacyanoferrate trihydrate and potassium dichromate in 0.1 M cacodylate for 1 hr, stained with 2% uranyl acetate in double distilled water for 1 hr, dehydrated in graded ethanol solutions and embedded in epoxy resin. Ultrathin sections (70-90 nm) were obtained with a Leica EMUC7 ultramicrotome and transferred to Formvar Support film slot grids (EMS). Grids were stained with lead citrate and examined with a Tecnai SPIRIT transmission electron microscope (Thermo Fisher Scientific). Digital electron micrographs were acquired with a bottom-mounted Gatan OneView camera.
FACs Sorting. Cells were isolated from Tg(pnp4a:PALM-mCherry) positive fish cells and sorted via Fluorescence-Activated Cell Sorting (FACS). Following resuspension in 5 ml cold HL-15 with 1% BSA, the isolated cells were incubated with Hoechst to mark the nuclei for 30 min prior to FACs. Cells were analyzed and sorted using a BD FACSAria™ III Cell Sorter with a 100 µM nozzle. Cells were illuminated using a both 405 and 561 nm lasers. Cells were gated based on attributes to separate cells from each other as well as from cellular debris. Cellular debris were detected using forward, side scatter and Hoechst signals to select against the smallest particles (1 mm or less). Cells were additionally sorted and enriched based on detection using 561 nm filters, corresponding to the pnp4a:PALM-mCherry signal. Cells were collected into ice-cold HL-15 medium with 1% BSA and kept on ice until mounted on grids for downstream imaging.
High pressure freezing zebrafish larvae. 56, 72 and 96 hpf zebrafish larvae were anesthetized using Tricaine and decapitated. About 3 larvae were placed between two metal discs (2 mm diameter; cavity, 0.2 mm) in 10% dextran solution and cryo-immobilized using a Leica LM ICE high pressure freezing device (Leica Microsystems, Germany).
Freeze fracture, cryo-SEM. The high-pressure frozen zebrafish larvae samples were shuttled using a vacuum cryo-transfer device (VCT 100, Leica Microsystems, Germany). The sample was transferred into a freeze-etching/freeze-fracture device (BAF 60, Bal-Tec, Germany), the stage of which was maintained at -120°C and a vacuum of about 5 × 10 −7 mbar. After fracturing, the disc remaining in the sample holder was coated with 6 nm of platinum. The coated sample was transferred to the SEM (Ultra 55, Zeiss, Germany) and observed at −120°C and a vacuum of about 5 × 10 −7 mbar, using an acceleration voltage of 1 kV, an aperture size of 10 μm, and a working distance of 2 mm.
Cryo FIB-SEM. High-pressure frozen larvae samples were fractured as done for the cryo SEM, except the temperature was at -160°C. The coated sample was transferred to cryo FIB-SEM (Crossbeam 550, Zeiss, Germany) using a vacuum cryo-transfer device (VCT 100, Leica Microsystems, Germany). At all times, the temperature of the cryo-stage was below −150°C, and the vacuum inside the chamber of was around 5 × 10 −7 mbar. Rough milling was done prior to imaging (in order to expose the region of interest) at 30 kV FIB acceleration voltage and 1.5 nA FIB probe current. Fine milling was done with the following milling parameters: 5 mm working distance, 30 kV FIB acceleration voltage and between 100 to 700 pA FIB probe current. A total of 5 data sets from different samples (56, 72 and 96 hpf frozen larvae) were milled that had dimensions of 30-65 µm × 8.3-25 mm with slice thickness of 10 or 20 nm. Imaging was done using the following parameters: lateral image pixel size between 6 and 19.5 nm, field of view 12-40 µm width and 2 kV SEM acceleration voltage.
Image processing of 3D data was performed as follows: To correct for the vertical stripes occurring in the stacks, we applied the wavelet decomposition algorithm as published in Spehner D. et al. 1 . We've used the coif wavelet of N = 10 and vertical coefficient σ = 10. This approach was chosen as it preserves the overall intensity of the image better than with a simple Fourier filtering approach. The images were then corrected for local charge imbalance using the morphological reconstruction (dilation) approach 1 . The algorithm was implemented using python 3.7. The images were automatically aligned with the MIB software (Microscopy Image Browser, University of Helsinki Institute of Biotechnology EM unit) 2 . The aligned stack was further aligned manually using the Amira3D (Thermo Scientific) alignment tool.
CryoET. Sorted iridophores cells (3.5 μL) with 15 nm gold beads (1 μL) were applied to glowdischarged holey carbon R2/2 Cu 200 SiO2 mesh grids (Quantifoil) coated with collagen, Type I, Rat Tail (EMD Millipore 08-115) for cell adherence. The grids were blotted and vitrified by plunging into liquid ethane using a Leica EM GP automatic plunger, under 4°C and 90% humidity conditions. Frozen grids were kept in liquid nitrogen until used. Data was collected on a Titan Krios TEM G3i (Thermo Fisher Scientific) equipped with a BioQuantum energy filter with a K3 direct electron detector (Gatan Inc.). Data sets were collected at 300 kV with the K3 camera (counting mode) using SerialEM software 3 . The TEM magnification corresponded to a camera pixel size of 1.6 Å, and the target defocus was set to 3 μm. The total dose for a full tilt series was 120 electrons per Å 2 . Tomograms tilt series were collected using the dose-symmetric scheme, ±60° at 2° degrees steps. The tilt series images alignment and reconstruction were performed in IMOD 4 .

Cryo 4D STEM. Scanning transmission electron microscope (STEM) images and analytical EDS maps were acquired in a double aberration-corrected Themis-Z microscope (Thermo Fisher
Scientific Electron Microscopy Solutions, Hillsboro, USA, (TFS)) at an accelerating voltage of 200 kV. All measurements were completed on fully vitrified plunge-frozen samples in a Gatan 914 cryo-transfer holder. STEM images were recorded using a Fischione Model 3000 detector and a TFS BF detector. EDS hyperspectral data was obtained with a Super-X SDD detector and quantified with the Velox software (TFS) through background subtraction and spectrum deconvolution.
4D STEM 5 datasets were obtained on the EMPAD detector (Electron Microscope Pixel Array Detector) 6 that allowed rapid data collection of unsaturated diffraction patterns with single electron sensitivity. An electron probe with a convergence angle of 0.2 mrad was adjusted and further defocussed by typically 5-10 µm to increase the real space probe size to several 10 nm in diameter and to reduce the electron fluence. The defocussed electron beam was rastered without beam overlap across the sample. A primary beam current between 1 pA resulted in a total fluence of less than 1 e -/Å 2 per exposure frame time of 2 ms, i.e., per beam position. All 4D STEM datasets were analyzed with custom-written software.
Segmentation-Segmentation and 3D representation of the reconstructed data was done using Amira® software (Thermo Scientific). Data segmentation was performed based on contrast variations following the unique shape and structure of each component.   Cells were isolated from Tg(pnp4a:PALM-mCherry) positive fish mix. After cell isolation, cells were analyzed and sorted using a BD FACSAria™ III Cell Sorter and were gated based on attributes to separate cells from each other as well as from cellular debris. Cellular debris were detected using forward, side scatter and Hoechst signals to select against the smallest particles (1 mm or less). Cells were additionally sorted and enriched based on detection using 561 nm filters, corresponding to the pnp4a:PALM-mCherry signal. Different populations of iridophores can be distinguished using this method based on their mCherry signal in combination with the intensity of their side scatter.